Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of ß-barrel pore-forming toxins (ß-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane ß-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif (CRM) in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

Virilising Adrenal Adenoma in Children.

ABSTRACT: Tumours of the adrenal cortex are most commonly seen in adults but rare in children. The clinical manifestations depend on the age and sex of patients; about two-thirds of the cases virilisation is the predominant presentation, whereas many presents with both virilisation and cushingoid features. Diagnosis is confirmed by hormonal evaluation and radiological investigations. Surgical removal is the mainstay of treatment. Usually, there is a good prognosis in paediatric patients, whereas the high mortality rate reported in older literature may have been due to big tumour size, post-operative complications and inadequate steroid replacement. Here, we are presenting a series of three cases during childhood with virilising features due to adrenocortical tumours. All of them presented predominantly with features suggestive of virilisation. All patients underwent surgery and had a good outcome despite big tumour size in one of the patients. Histology revealed a benign lesion in the form of adrenal adenoma. Most virilisation and cushingoid features disappeared in the follow-up (median - 1 year). Although these tumours are rare, a high index of suspicion should be kept in children with cushingoid features, virilisation or a combination of both of them. Even if the tumour size is big, adequate steroid replacement and supportive management postoperatively have led to good prognosis in our patients.

Vibrio parahaemolyticus thermostable direct haemolysin induces non-classical programmed cell death despite caspase activation.

Thermostable direct haemolysin (TDH) is the key virulence factor secreted by the human gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin. It evokes potent cytotoxicity, the mechanism of which still remains under-explored. Here, we have elucidated the mechanistic details of cell death response elicited by TDH. Employing Caco-2 intestinal epithelial cells and THP-1 monocytic cells, we show that TDH induces some of the hallmark features of apoptosis-like programmed cell death. TDH triggers caspase-3 and 7 activations in the THP-1 cells, while caspase-7 activation is observed in the Caco-2 cells. Interestingly, TDH appears to induce caspase-independent cell death. Higher XIAP level and lower Smac/Diablo level upon TDH intoxication provide plausible explanation for the functional inability of caspases in the THP-1 cells, in particular. Further exploration reveals that mitochondria play a central role in the TDH-induced cell death. TDH triggers mitochondrial damage, resulting in the release of AIF and endonuclease G, responsible for the execution of caspase-independent cell death. Among the other critical mediators of cell death, ROS is found to play an important role in the THP-1 cells, while PARP-1 appears to play a critical role in the Caco-2 cells. Altogether, our work provides critical new insights into the mechanism of cell death induction by TDH, showing a common central theme of non-classical programmed cell death. Our study also unravels the interplay of crucial molecules in the underlying signalling processes. Our findings add valuable insights into the role of TDH in the context of the host-pathogen interaction processes.

The changing landscape of interventional cardiology: A survival guide in the era of health system consolidation: The Society of Cardiovascular Angiography and Interventions affirms the value of this manuscript.

Practice environments for interventional cardiologists have evolved dramatically and now include small independent practices, large cardiology groups, multispecialty groups, and large integrated health systems. Increasingly, cardiologists are employed by hospitals or health systems. Data from MedAxiom and the American College of Cardiology (ACC) demonstrate an exponential increase in the percentage of cardiologists in employed positions from 10% in 2009 to 87% in 2020. This white paper explores these profound changes, considers their impact on interventional cardiologists, and offers guidance on how interventional cardiologists can best navigate this challenging environment. Finally, the paper offers a potential model to improve the employed physician experience through greater physician involvement in decision making, which may increase jobs satisfaction.

Advocacy 101 for Interventional Cardiologists: A Society for Cardiovascular Angiography & Interventions Policy Statement.

Advocacy is a core mission of the Society for Cardiovascular Angiography & Interventions (SCAI). SCAI advocates on behalf of interventional cardiologists and our patients. This document provides foundational information and a toolkit for grassroots advocacy by interventional cardiologists. The first half of the document summarizes how health care laws are made, how medical devices are approved, and how procedure reimbursement is determined. The second half of the document is a playbook of advocacy strategies: legislative advocacy, judicial advocacy, advocacy with regulators and payors, advocacy in the media, and participation in SCAI advocacy initiatives, such as the Government Relations Committee and SCAI Political Action Committee. Equipped with this toolbox, interventional cardiologists must increase our advocacy activities with government, payors, and industry.

Role of Laparoscopic Transillumination Guidance During Hysteroscopic Metroplasty in Simplifying Surgical Management of Type II Robert's Uterus.

Robert's uterus is a rare variant of septate uterus with an asymmetrical septum which divides the uterine cavity into a noncommunicating hemiuterus causing hematometra and other communicating hemiuterus with a single cervix and a normal fundal contour (U2bC3V4 ESHRE classification). It is a cause of severe dysmenorrhea in young girls. However, there is a type of Robert uterus (Type II) which does not have collection in the blind cavity and causes symptoms later, similar to our case. We describe a case of hysteroscopic septum resection (metroplasty) with laparoscopic guidance by transillumination in a case of Type II Robert's uterus in a 25-year-old nulliparous woman. Thick muscular septum posed a surgical challenge which was supplemented by astutely utilizing laparoscopic transillumination.

Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient ß-barrel pore formation.

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging ß-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane ß-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane ß-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric ß-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional ß-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.

Pore formation-independent cell death induced by a ß-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin (ß-PFT). It exhibits potent hemolytic activity against erythrocytes that appears to be a direct outcome of its pore-forming functionality. However, VCC-mediated cell-killing mechanism is more complicated in the case of nucleated mammalian cells. It induces apoptosis in the target nucleated cells, mechanistic details of which are still unclear. Furthermore, it has never been explored whether the ability of VCC to trigger programmed cell death is stringently dependent on its pore-forming activity. Here, we show that VCC can evoke hallmark features of the caspase-dependent apoptotic cell death even in the absence of the pore-forming ability. Our study demonstrates that VCC mutants with abortive pore-forming hemolytic activity can trigger apoptotic cell death responses and cytotoxicity, similar to those elicited by the wild-type toxin. VCC as well as its pore formation-deficient mutants display prominent propensity to translocate to the target cell mitochondria and cause mitochondrial membrane damage. Therefore, our results for the first time reveal that VCC, despite being an archetypical ß-PFT, can kill target nucleated cells independent of its pore-forming functionality. These findings are intriguing for a ß-PFT, whose destination is generally expected to remain limited on the target cell membranes, and whose mode of action is commonly attributed to the membrane-damaging pore-forming ability. Taken together, our study provides critical new insights regarding distinct implications of the two important virulence functionalities of VCC for the V. cholerae pathogenesis process: hemolytic activity for iron acquisition and cytotoxicity for tissue damage by the bacteria.

Cryo-EM elucidates mechanism of action of bacterial pore-forming toxins.

Pore-forming toxins (PFTs) rupture plasma membranes and kill target cells. PFTs are secreted as soluble monomers that undergo drastic structural rearrangements upon interacting with the target membrane and generate transmembrane oligomeric pores. A detailed understanding of the molecular mechanisms of the pore-formation process remains unclear due to limited structural insights regarding the transmembrane oligomeric pore states of the PFTs. However, recent advances in the field of cryo-electron microscopy (cryo-EM) have led to the high-resolution structure determination of the oligomeric pore forms of diverse PFTs. Here, we discuss the pore-forming mechanisms of various PFTs, specifically the mechanistic details contributed by the cryo-EM-based structural studies.

Study of effect of gluten-free diet on vitamin D levels and bone mineral density in celiac disease patients.

Objective: Celiac disease (CD) is a multifactorial immune-mediated enteropathy caused by a response to ingested gluten. The current available treatment for CD is lifelong gluten-free diet (GFD). This study was done to see the effect of GFD on Vitamin D levels and bone mass density in celiac patients. Methods: A prospective interventional study on newly diagnosed celiac patients was conducted in the Pediatrics department of a tertiary care teaching institute in 2 stages viz. on presentation and after 6 months of GFD. Anthropometric measurements, biochemical investigations, Vitamin D levels, and DEXA scan was done at recruitment and after 6 months of GFD and was analyzed. Results: In newly diagnosed 60 pediatric celiac patients, positive effect of GFD on anthropometry, hemoglobin, Vitamin D levels, DEXA scan parameters was observed. Significant difference was found in Vitamin D levels which increased from baseline 14.85 ± 5.39 to 18.22 ± 5.67 ng/ml after 6 months of GFD (P < 0.05). Significant difference was found in BMD (mean Z-score) which increased from -0.941 ± 0.738 to -0.640 ± 0.60 after 6 months of GFD (P < 0.001). Conclusion: Our study concluded that there is significant increase in vitamin D levels as well as Z-score, bone mass density (BMD) and bone Mass Content (BMC) after 6 months of GFD.

Membrane Dynamics and Remodelling in Response to the Action of the Membrane-Damaging Pore-Forming Toxins.

Pore-forming protein toxins (PFTs) represent a diverse class of membrane-damaging proteins that are produced by a wide variety of organisms. PFT-mediated membrane perforation is largely governed by the chemical composition and the physical properties of the plasma membranes. The interaction between the PFTs with the target membranes is critical for the initiation of the pore-formation process, and can lead to discrete membrane reorganization events that further aids in the process of pore-formation. Punching holes on the plasma membranes by the PFTs interferes with the cellular homeostasis by disrupting the ion-balance inside the cells that in turn can turn on multiple signalling cascades required to restore membrane integrity and cellular homeostasis. In this review, we discuss the physicochemical attributes of the plasma membranes associated with the pore-formation processes by the PFTs, and the subsequent membrane remodelling events that may start off the membrane-repair mechanisms.

SCAI Publications Committee Manual of Standard Operating Procedures: 2022 Update.

Evidence-based recommendations for clinical practice are intended to help health care providers and patients make decisions, minimize inappropriate practice variation, promote effective resource use, improve clinical outcomes, and direct future research. SCAI has been engaged in the creation and dissemination of clinical guidance documents since the 1990s. These documents are a cornerstone of the Society's education, advocacy, and quality improvement initiatives. The Publications Committee is charged with the oversight of SCAI's clinical documents program and has published the first iteration of this manual of standard operating procedures in 2019 to ensure consistency, methodological rigor, and transparency in the development and endorsement of the Society's documents. The manual has been updated based on feedback from the implementation of the original version to add specificity and expand the breadth of available document formats. The manual is intended for reference by the Publications Committee, document writing groups, external collaborators, SCAI representatives, peer reviewers, and anyone seeking information about the SCAI documents program.

Knowledge, Attitude and Perception of HIV/Aids Among Antenatal Women and its Correlation with their Socio-Demographic Profile: Study from a Tertiary Care Centre of Northern India.

PURPOSE OF STUDY: To evaluate the knowledge, attitude and perception of HIV/AIDS among antenatal women and to correlate them with their socio-demographic profile. METHODS: We conducted this study on 400 pregnant women attending the antenatal clinic of our hospital for the first time irrespective of their period of gestation, age and parity. All the participants were interviewed with the help of a predesigned questionnaire which included their socio-demographic details and questions to assess their knowledge and attitude toward HIV/AIDS. Data were analyzed using SPSS version 22 and expressed in the form of percentage, frequency distribution, mean, standard deviation and p value. RESULTS: Antenatal women of the study population were having unsatisfactory knowledge about HIV/AIDS and prevention of MTCT. 26% women were totally unaware of any entity like HIV. 44% participants did not know the most common way of spread of HIV. Only half of the subjects knew the correct preventive measures for HIV/AIDS. 54% knew about MTCT, but only 24% knew about its transmission through breast milk. Knowledge and attitude was found to be significantly improving with socioeconomic status. CONCLUSION: Indian antenatal women have poor awareness and wrong perception about HIV/AIDS and its mother to child transmission (MTCT). Targeted educational interventions can be formulated to increase awareness among antenatal women about prevention of vertical transmission of HIV.

Intraoperative Dislodgment and Retrieval of Broken Parts of Laparoscopic Instruments: Arduous Exercise and Lessons Gleaned.

BACKGROUND: Dislodgement and breakage of instruments in laparoscopy is a rare event which not only surmounts the anxiety of the team, but also imposes an exceedingly onerous situation for the patient. Frequently a broken fragment of an instrument is confined to an area remote from the primary operative site and gets entrapped in the bowel loops or in the omentum. METHOD: We present the intraoperative loss of the distal tip of three 5-mm laparoscopy instruments (monopolar L-hook, myoma screw and tenaculum) in the abdominal cavity during endoscopy. RESULT: Various retrieval methods for laparoscopy instruments have been described. CONCLUSION: The distal working tips of laparoscopic instruments have delicate functioning and tend to fall off or break during usage. Maintenance of instruments used in endoscopy requires special care and should be done as outlined by the manufacturer. Reporting of such incidents should be encouraged and published despite the discomposure accompanying it as it aids in better understanding and learning to handle these situations.

Pore-forming toxins in infection and immunity.

The integrity of the plasma membranes is extremely crucial for the survival and proper functioning of the cells. Organisms from all kingdoms of life employ specialized pore-forming proteins and toxins (PFPs and PFTs) that perforate cell membranes, and cause detrimental effects. PFPs/PFTs exert their damaging actions by forming oligomeric pores in the membrane lipid bilayer. PFPs/PFTs play important roles in diverse biological processes. Many pathogenic bacteria secrete PFTs for executing their virulence mechanisms. The immune system of the higher vertebrates employs PFPs to kill pathogen-infected cells and transformed cancer cells. The most obvious consequence of membrane pore-formation by the PFPs/PFTs is the killing of the target cells due to the disruption of the permeability barrier function of the plasma membranes. PFPs/PFTs can also activate diverse cellular processes that include activation of the stress-response pathways, induction of programmed cell death, and inflammation. Upon attack by the PFTs, host cells may also activate pathways to repair the injured membranes, restore cellular homeostasis, and trigger inflammatory immune responses. In this article, we present an overview of the diverse cellular responses that are triggered by the PFPs/PFTs, and their implications in the process of pathogen infection and immunity.

Laparoscopic Excision of Cesarean Scar Ectopic Pregnancy.

BACKGROUND: Cesarean scar ectopic pregnancies are increasing in frequency, due to rise in cesarean deliveries. They should be managed early in pregnancy, preferably by surgical excision, failing which they may rupture, or later develop into morbidly adherent placenta. METHODS: This is a series of five cases described to explain the instrumentations and techniques in the laparoscopic excision of cesarean scar ectopic pregnancies. Written consent was taken from the patients. RESULTS: All five patients underwent successful laparoscopic excision. Follow-up period was uneventful. CONCLUSION: Laparoscopic excision of cesarean scar ectopic is a technically demanding procedure, but with excellent results. All gynecologists should be familiar with this technique due to the increasing incidence of cesarean scar ectopic gestations.